Clevidipine emulsion formulations containing antimicrobial agents

ABSTRACT

Pharmaceutical formulations comprising clevidipine in an oil-in-water formulation that is resistant to microbial growth and stable against the formation of impurities.

This application claims priority from U.S. provisional application No.61/392,294 filed on Oct. 12, 2010 and U.S. Nonprovisional applicationSer. No. 13/270,004 filed Oct. 10, 2011. The above referencedapplication is incorporated by reference herein as if set forth in itsentirety.

FIELD OF THE INVENTION

The instant invention relates to an oil-in-water clevidipine emulsionformulation that is stable and resistant to microbial growth.

BACKGROUND OF THE INVENTION

Clevidipine is a dihydropyridine calcium channel blocker that reducesblood pressure in a subject to which it is administered. It ischaracterized as a short-acting, highly selective drug that is typicallyused in a hospital setting due to its rapid metabolism, exhibiting aninitial phase half-life of about one minute and a terminal half-life ofapproximately 15 minutes. More detailed information on short-actingdihydropyridines can be found in U.S. Pat. No. 5,856,346, the entiredisclosure of which is incorporated by reference herein as if set forthin its entirety.

Clevidipine is further characterized by having low solubility in waterand moderate to high solubility in lipids. When clevidipine is dissolvedin oil-in-water emulsions, it results in better solubility and/or lessside effects than when other conventional solution formulations areutilized. Cleviprex® is a clevidipine oil-in-water emulsion formulationapproved by the U.S. FDA for intravenous administration in the treatmentof acute hypertension, primarily in the emergency room and intensivecare unit, and in pre-, peri-, and post-operative settings.

Cleviprex® can support microbial growth since it contains soybean oiland egg yolk phospholipids. Therefore, clevidipine oil-in-water emulsionformulations require strict aseptic technique during handling andadministration to avoid microbial contamination that can causeinfections among patients. To minimize the possibility of microbialcontamination, it is recommended that such formulations be discardedfour hours after opening. Such a requirement places a burden on healthcare providers in that fresh vials of the drug must be continuallyobtained and set up while the patient is being treated. Accordingly,there exists a need for a stable clevidipine emulsion formulation thatis resistant to microbial growth, thereby diminishing the risk ofmicrobial contamination in patients and providing greater ease inhandling. Such a formulation would also result in cost savings to thehealth care providers and patients in decreasing the wastage ofclevidipine and reducing the time-consuming efforts involved withmanipulation and replacement of vials containing the drug.

SUMMARY OF THE INVENTION

Based on the state of the art, it is the object of the present inventionto provide a clevidipine emulsion formulation that is not only stableagainst formation of impurities but having a reduced propensity formicrobial contamination.

In a first embodiment of the present invention, there is provided apharmaceutical formulation comprising clevidipine, or a pharmaceuticallyacceptable salt or ester thereof, an antimicrobial agent, a lipid, anemulsifier, a tonicity modifier and water.

In one aspect of this embodiment, the present invention provides anoil-in-water emulsion wherein clevidipine is dispersed or dissolved in alipid, such as soy bean oil, which in turn is emulsified with egg yolkphospholipids in water. The tonicity of emulsion is adjusted withglycerin and further comprises an amount of EDTA sufficient to inhibitgrowth of microorganisms in the formulation in the event of accidentalextrinsic contamination.

In second aspect of this embodiment, the present invention provides anoil-in-water emulsion wherein clevidipine is dispersed or dissolved in alipid, such as soy bean oil, which in turn is emulsified with egg yolkphospholipids in water. The tonicity of emulsion is adjusted withglycerin and further comprises an amount of EDTA and sodium citratesufficient to inhibit growth of microorganisms in the formulation in theevent of accidental extrinsic contamination.

In a second embodiment of the invention, there is provided apharmaceutical formulation comprising clevidipine, or a pharmaceuticallyacceptable salt or ester thereof, an antimicrobial agent, a lipid, anemulsifier, a co-emulsifier, a tonicity modifier and water.

In one aspect of this embodiment, the present invention provides anoil-in-water emulsion wherein clevidipine is dispersed or dissolved in alipid, such as soy bean oil, which in turn is emulsified with egg yolkphospholipids in water, and wherein the emulsion is further stabilizedby the co-emulsifier oleic acid. The tonicity of emulsion is adjustedwith glycerin and further comprises an amount of EDTA sufficient toinhibit growth of microorganisms in the formulation.

In a second aspect of this embodiment, the present invention provides anoil-in-water emulsion wherein clevidipine is dispersed or dissolved in alipid, such as soy bean oil, which in turn is emulsified with egg yolkphospholipids in water, and wherein the emulsion is further stabilizedby the co-emulsifier oleic acid. The tonicity of emulsion is adjustedwith glycerin and further comprises an amount of EDTA and sodium citratesufficient to inhibit growth of microorganisms in the formulation in theevent of accidental extrinsic contamination.

In a third embodiment of the invention, there is provided apharmaceutical formulation comprising clevidipine, or a pharmaceuticallyacceptable salt or ester thereof, an antimicrobial agent, anantioxidant, a lipid, an emulsifier, a tonicity modifier and water.

In one aspect of this embodiment, the present invention provides anoil-in-water emulsion wherein clevidipine is dispersed or dissolved in alipid, such as soy bean oil, which in turn is emulsified with egg yolkphospholipids in water and stabilized by the antioxidant sodiumascorbate. The tonicity of emulsion is adjusted with glycerin andfurther comprises an amount of EDTA sufficient to inhibit growth ofmicroorganisms in the formulation.

In a fourth embodiment of the invention, there is provided apharmaceutical formulation comprising clevidipine, or a pharmaceuticallyacceptable salt or ester thereof, an antimicrobial agent, anantioxidant, a lipid, an emulsifier, a co-emulsifier, a tonicitymodifier and water.

In one aspect of this embodiment, the present invention provides anoil-in-water emulsion wherein clevidipine is dispersed or dissolved in alipid, such as soy bean oil, which in turn is emulsified with egg yolkphospholipids in water and wherein the emulsion is further physicallystabilized by the co-emulsifier oleic acid and chemically stabilized bythe antioxidant sodium ascorbate. The tonicity of emulsion is adjustedwith glycerin and further comprises an amount of EDTA sufficient toinhibit growth of microorganisms in the formulation.

In aspects of embodiments where the formulation comprises anantioxidant, the antioxidant reduces the formation of oxidativedegradants H324/78 and H152/66.

In preferred aspects of each embodiment of the invention, theantimicrobial agent is an alcohol or a chelating agent. More preferably,the antimicrobial agent is disodium edetate (EDTA) or sodium citrate, orboth.

In preferred aspects of each embodiment of the invention, thepharmaceutical formulation is sterile. In the event of accidentalcontamination, the pharmaceutical formulation will retard the growth ofmicroorganisms to no more than 1 log within at least about 24 hours.

In each embodiment of the invention, clevidipine and the emulsionmaintain their stability in the formulation. In each embodiment of theinvention, the pharmaceutical formulation is for parenteraladministration. In each embodiment of the invention, the pharmaceuticalformulation will have a pH that ranges from about 6.0 to about 8.8.

BRIEF DESCRIPTION OF THE FIGURES

Understanding of the present invention will be facilitated byconsideration of the following detailed description of the embodimentsof the present invention taken in conjunction with the accompanyingdrawings:

FIG. 1: clevidipine degradation pathway.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that the figures and descriptions of the presentinvention have been simplified to illustrate elements that are relevantfor a clear understanding of the present invention, while eliminating,for the purpose of clarity, many other elements found in typicalpharmaceutical compositions and methods of stabilization. Those ofordinary skill in the art will recognize that other elements and/orsteps are desirable and/or required in implementing the presentinvention. However, because such elements and steps are well known inthe art, and because they do not facilitate a better understanding ofthe present invention, a discussion of such elements and steps is notprovided herein. The disclosure herein is directed to all suchvariations and modifications to such elements and methods known to thoseskilled in the art. Furthermore, the embodiments identified andillustrated herein are for exemplary purposes only, and are not meant tobe exclusive or limited in their description of the present invention.

As used herein, the term “clevidipine” shall mean and include allvarieties or forms of clevidipine. Unless otherwise specified, examplesof such forms include all pharmaceutically acceptable salts, esters,isomers, stereo isomers, crystalline and amorphous forms. One particularexample is clevidipine butyrate. The amount of clevidipine in theformulations of the present invention can vary depending on the totaloverall volume of the formulation and the concentration of the othercomponents. However, the amount of clevidipine in the formulation willgenerally range from about 0.005 to about 1.0% w/v, and includes rangesof from about 0.03 to about 0.5% w/v, and from about 0.01 to about 1.0%w/v. In particular embodiments, the amount of clevidipine in theformulation will be about 0.05, 0.1, or 0.3% w/v.

As used herein, the term “pharmaceutically acceptable salt” shall referto salts prepared from pharmaceutically acceptable non-toxic bases oracids including inorganic or organic bases and inorganic or organicacids. Examples of salts derived from inorganic bases include aluminum,ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganicsalts, manganous, potassium, sodium, zinc, and the like. Particularlypreferred are the ammonium, calcium, magnesium, potassium, and sodiumsalts. Salts derived from pharmaceutically acceptable organic non-toxicbases include salts of primary, secondary, and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines, and basic ion exchange resins, such as arginine, betaine,caffeine, choline, N,N′-dibenzylethylenediamine, diethylamine,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethyl-morpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperidine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylamine,tripropylamine, tromethamine, and the like.

As used herein, the term “pharmaceutically acceptable ester” shall referto esters prepared by reaction of an oxo-acid with a compound containinga hydroxyl group. Generally esters are derived from an inorganic ororganic acid and an alcohol. More commonly esters are prepared bycondensing an organic acid and an alcohol. Examples of suitable estersthat may be used in the formulations of the present invention includebutyric acid esters, such as those prepared in accordance with teachingsin U.S. Pat. Nos. 5,856,346, 5,739,152, 6,350,877 and the like.

Clevidipine is manufactured by reaction of4-(2′,3′-dichlorophenyl)-1,4-dihydro-5-methoxycarbonyl-2,6-dimethyl-3-pyridinecarboxylicacid with chloromethyl butyrate to obtain clevidipine. This reaction canbe done optionally in the presence of a corresponding hydrogencarbonate, such as KHCO₃, in refluxing acetonitrile. Inorganic salts canbe removed by filtration and the product is crystallized by the additionof isopropanol and water with subsequent cooling. It can also becrystallized by exchanging solvent from acetonitrile to a mixture ofalcohol, such as ethanol or isopropanol, and water with repeatedevaporations. In the further purification of the product the crystalsare washed with a mixture of water and ethanol or isopropanol. Theproduct can be dissolved in refluxing isopropanol, crystallized bycooling, isolated by filtration and finally washed with a water andisopropanol mixture. A more detailed description of the manufacturingprocess of clevidipine can be found in U.S. Pat. No. 6,350,877, theentire disclosure of which is incorporated by reference herein as if setforth in its entirety. As used herein, the range of clevidipine that maybe used in the forulations includes a range of from about 0.005% toabout 1% w/v.

Compositions containing clevidipine as an active ingredient aresensitive to water, heat, and light. Clevidipine degrades under adverseconditions into several impurities that compromise the potency ofclevidipine. A degradation pathway of clevidipine is shown in FIG. 1.This pathway identifies a number of clevidipine degradation products,including for example, H152/81, H168/79, H207/59, H324/78 and H152/66.

As with any of the exemplary embodiments of pharmaceutical compositionsdescribed herein, it is preferred that the level of clevidipineimpurities in the composition is as low as possible. Therefore, whilevarious exemplary embodiments of pharmaceutical compositions includelevels of impurities within acceptable and effective ranges for thecompositions as a whole, the more pure the composition, the moredesirable the composition. The amount of clevidipine degradationproducts present in the formulations of the present invention is lessthan about 5%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, or0.2%, or even less.

The pharmaceutical formulations of the present invention are emulsionformulations. For compounds that have poor solubility and stability inwater, emulsions offer much better solubility, and stability thanconventional aqueous solutions. Oil-in-water emulsions also prevent thecompound from adherence to plastic infusion sets that are to be usedwhen administering the compound. Further information regarding theformulation of clevidipine can be found in U.S. Pat. No. 5,739,152, theentire disclosure of which is incorporated by reference herein as if setforth in its entirety.

As used herein, the term “antimicrobial agent” means an agent thatinhibits the growth of microorganisms such as bacteria and fungi (moldsand yeast). Particular classes of antimicrobial agents include chelatingagents and alcohols. Chelating agents include, but are not limited to,ethylenediamine tetraacetic acid (EDTA) and salts thereof, citric acidand salts there of, and the like. Exemplary chelating agents aredisodium edetate and sodium citrate. Alcohols include, but are notlimited to, benzyl alcohol and chlorobutanol. Representativeantimicrobial agents include EDTA, ascorbic acid, BHA/BHT, benzylalcohol, benzoic acid, citric acid, edetic acid, parabens, phenol,propyl gallate, sorbic acid, sodium bisulfite, sodium sulfite, benzoicacid, chlorobutanol, chlorocresol, cresol, dehydroacetic acid, phenol,potassium benzoate, potassium sorbate, sodium benzoate, sodiumdehydroacetate, sodium propionate, sorbic acid, thymol, benzalkoniumchloride, benzethonium chloride, butyl paraben, cetylpyridiniumchloride, ethylparaben, methylparaben, methylparaben sodium,propylparaben, propylparaben sodium, chlorocresol, cresol, dehydroaceticacid, ethylparaben, methylparaben, methylparaben sodium, phenol,potassium sorbate, thimersol and the various salt forms for thesecompounds.

In general the term “EDTA” means ethylenediamine tetraacetic acid andits derivatives and salts thereof. Particular derivative of use in thepresent invention includes disodium edetate. The nature of the EDTAderivative or salt is not critical, provided that it inhibits growth ofmicroorganisms in the formulations of the present invention.

The amount of antimicrobial agent in the formulations of the presentinvention can vary depending on the total overall volume of theformulation and the concentration of the other components. However, theamount of the antimicrobial agent in the formulation will generallyrange from about 0.001 to about 1.5% w/v, and includes from about 0.005to about 0.5% w/v, from about 0.001 to about 0.1% w/v, from about 0.01to about 0.1% w/v, and from about 0.1 to about 1% w/v. In particularembodiments, the amount of the antimicrobial agent in the formulationwill be about 0.1, 0.2, or 0.4% w/v. Where a chelating agent is used asthe antimicrobial agent, the amount of chelating agent in theformulation will generally range from about 0.001% to about 0.5% w/v.The antimicrobial agent may comprise one, or more than one agent, suchas two, three, four, etc., different antimicrobial agents.

As used herein, the term “antioxidant” in the formulations includessodium ascorbate, cysteine hydrochloride, sodium bisulfite, sodiummetabisulfite, sodium sulfite, ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate,tocopherol, and their pharmaceutically acceptable salts. An exemplaryantioxidant is sodium ascorbate. The amount of antioxidant in theformulations of the present invention can vary depending on the totaloverall volume of the formulation and the concentration of the othercomponents. However, the amount of the antioxidant in the formulationwill generally range from about 0.01 to about 1.0% w/v, and includesfrom about 0.05 to about 1.0% w/v, and from about 0.05 to about 0.5%w/v. In particular embodiments, the amount of antioxidant in theformulation will be about 0.1% w/v.

As used herein, the term “lipid” in the formulations is anypharmaceutically acceptable oil, preferably a triglyceride such assoybean oil, safflower seed oil, olive oil, cottonseed oil, sunfloweroil, sesame oil, peanut oil, corn oil, a medium chain triglyceride (suchas Miglyol™ 812 or 810) or triacetin. The lipid may also be a propyleneglycol diester or monoglyceride (such as acetylareal monoglyceride). Thelipid can also be a mixture of one or more of these lipids. An exemplarylipid is soybean oil. The amount of lipid in the formulations of thepresent invention can vary depending on the total overall volume of theformulation and the concentration of the other components. However, theamount of lipid in the formulation will generally range from about 2 toabout 30% w/v, and includes from about 5 to about 30% w/v, and fromabout 10% to about 20% w/v. In particular embodiments, the amount oflipid in the formulation will be about 20% w/v.

As used herein, the term “emulsifier” represents suitablepharmaceutically acceptable surfactants used in the formulations,preferably naturally occurring phospholipids extracted from egg yolk orsoy bean, synthetic phosphatidyl cholines or purified phosphatidylcholines from vegetable origin. Hydrogenated derivatives can also beused, such as phosphatidyl choline hydrogenated (egg) and phosphatidylcholine hydrogenated (soya). The amount of phospholipid emulsifier inthe formulations of the present invention can vary depending on thetotal overall volume of the formulation and the concentration of theother components. However, the amount of emulsifier in the formulationwill generally range from about 0.2 to about 2.0% w/v, and includes fromabout 0.5 to about 1.5% w/v. In particular embodiments, the amount ofemulsifier in the formulation will be about 1.2% w/v.

As used herein, the term “co-emulsifier” represents a secondpharmaceutically acceptable surfactant that may be included in theformulations of the invention. Such surfactants include syntheticnonionic surfactants such as poloxamers (for example Poloxamer 188 and407), Cremophor™, poloxamines, polyoxyethylene stearates,polyoxyethylene sorbitan fatty acid esters or sorbitan fatty acidesters, derivatives of tocopherol such as tocopherol PEG succinate, longchain fatty acids such as oleic acid, stearic acid, palmitic acid, bileacids such as cholic acid and deoxycholic acid or surface activederivatives, and pharmaceutically acceptable salts thereof Δn exemplarysurfactant is oleic acid. The amount of co-emulsifier in theformulations of the present invention, when present, can vary dependingon the total overall volume of the formulation and the concentration ofthe other components. However, the amount of surfactant in theformulation will generally range from about 0.005 to about 2% w/v, andincludes from about 0.01 to about 2% w/v, and from about 0.01 to about1.0% w/v. In particular embodiments, the amount of surfactant in theformulation will be about 0.03% w/v.

As used herein, the term “tonicity modifier” in the formulations of thepresent invention includes sodium chloride, potassium chloride,mannitol, sucrose, lactose, fructose, maltose, dextrose, dextroseanhydrous, propylene glycol, glycerol and glycerin. An exemplarytonicity modifier is glycerin. The amount of tonicity modifier in theformulations of the present invention can vary depending on the totaloverall volume of the formulation and the concentration of the othercomponents. However, the amount of tonicity modifier in the formulationwill generally range from about 2 to about 3% w/v, and includes fromabout 2 to about 2.75% w/v. In particular embodiments, the amount oftonicity modifier in the formulation will be about 2.25% w/v. The terms“tonicity modifier” and “isotonicity adjuster” are used hereininterchangably.

The amount of water in the formulations of the present invention, suchas water-for-injections, is used to make up the volume to 100% w/v andcan vary depending on the total overall volume of the formulation andthe concentration of the other components.

As the formulations are intended for parenteral administration, theskilled artisan will understand that one or more additional componentsused in parenteral formulations may be included. Such additionalcomponents include stabilizing agents (e.g. carbohydrates, amino acidsand polysorbates, such as 5% dextrose), solubilizing agents (e.g.cetrimide, sodium docusate, glyceryl monooleate, polyvinylpyrolidone(PVP) and polyethylene glycol (PEG), buffers (e.g. acetates, citrates,phosphates, tartrates, lactates, succinates, amino acids and the like),preservatives (e.g. BHA, BHT, gentisic acids, vitamin E, ascorbic acid,sodium ascorbate and sulfur containing agents such as sulfites,bisulfites, metabisulfites, thioglycerols, thioglycolates and the like),suspending or viscosity agents, chelating agents, and administrationaids (e.g. local anesthetics, anti-inflammatory agents, anti-clottingagents, vaso-constrictors for prolongation and agents that increasetissue permeability).

Parenteral modes of administration include without limitation,intradermal, subcutaneous (s.c., s.q., sub-Q, Hypo), intramuscular(i.m.), intravenous (i.v.), intraperitoneal (i.p.), intra-arterial,intramedulary, intracardiac, intra-articular (joint), intrasynovial(joint fluid area), intracranial, intraspinal, and intrathecal (spinalfluids). Any known device useful for parenteral injection or infusion ofdrug formulations can be used to effect such administration.

In intravenous use, a sterile formulation of the present invention canbe dissolved or suspended in any of the commonly used sterileintravenous fluids and administered by infusion. Intravenous fluidsinclude, without limitation, physiological saline, phosphate bufferedsaline, 5% dextrose in water or Ringer's™ solution. The parenteraldosage form of formulations of the present invention can also be aready-to-use solution in sterile sealed vials, hermetically sealedampoules or in sterile pre-filled syringes, for example.

The sterile pre-filled syringes are syringes that contain a unit dose ofa pharmaceutical formulation of the present invention. Suitable syringesare widely available and well known to the skilled artisan. An exemplarysterile pre-filled syringe is one that has been loaded with a unit doseof the pharmaceutical formulation and that is enclosed in an opaque,sealed package from which oxygen has been excluded. For example, oxygenmay be displaced with CO₂ and/or N₂.

The pharmaceutical formulations of the present invention will have a pHthat ranges from about 6.0 to about 8.8. In particular embodiments, thepH ranges from about 6.5 to about 8.0. In particular embodiments, the pHis 6.2, 6.5, 6.75, 7.0, or 7.5. Bases such as NaOH, KOH, and Ca(OH)₂ maybe used to achieve the desired pH.

As used herein, the terms “inhibit”, “inhibiting” and “inhibition” havetheir ordinary and customary meanings, and include inhibiting the growthof a bacteria or fungus in the formulations of the present invention.Such inhibition may be described as no more than about 10 fold growthfor at least 24 hours following a low level (1-1000 Cfu/mL) of extrinsiccontamination. Such growth may be determined, for example, bydetermining the number of colony forming units in the formulation whencultured at room temperature.

The duration of time over which inhibition of microbial growth ismaintained will vary depending on the environmental conditions to whichthe formulation is exposed, e.g., the conditions under which a sterilevial of the formulation is pierced by a needle or sterility is otherwisebreached. However, in one embodiment of the invention, microbial growthis inhibited for at least about 24 or more hours after the formulationis exposed to low level extrinsic microbial contamination.

The skilled artisan will understand that the pharmaceutical formulationsof the present invention may be prepared using art-accepted means forpreparing emulsion formulations. A general procedure for preparingclevidipine formulations is described as follows: an oil phasecontaining soybean oil, clevidipine and egg yolk phospholipids is mixedwith an aqueous phase containing glycerin, at approximately 70° C. toform a coarse emulsion. The pH of the coarse emulsion is adjusted usingsodium hydroxide. Following pH adjustment, the coarse emulsion ishomogenized under high pressure to produce a fine particle size and thusa stable emulsion. The emulsion is filled into appropriate containersand sterilized in an autoclave.

The invention is described in greater detail by reference to thefollowing non-limiting examples.

EXAMPLES 1-5 Example 1

Formulations containing antimicrobial agents as shown in Table 1 belowwere are prepared by adding stock solutions of anti microbial agent atappropriate concentration to clevidipine emulsions.

TABLE 1 (% w/v) Function Example 1 Example 2 Example 3 Example 4 Example5 Active Clevidipine 0.05 0.05 0.05 0.05 0.05 ingredient butyrate Oilphase Soybean oil 20 20 20 20 20 Isotonicity Glycerin 2.25 2.25 2.252.25 2.25 adjuster Microbial Disodium 0 0.005 0.01 0 0.005 GrowthEdetate Retardant Microbial Benzyl 0 0 0 0.4 0.1 Growth AlcoholRetardant Emulsifier Purified egg 1.2 1.2 1.2 1.2 1.2 yolk phospholipidspH adjuster Sodium q.s q.s q.s q.s q.s hydroxide Aqueous Water for q.sto q.s to q.s to q.s to q.s to Phase Injection 100% 100% 100% 100% 100%

Sterile clevidipine oil-in-water compositions prepared as describedabove were evaluated for microbial growth. Microbial growth inhibitionwas tested using standard UPS test organisms. Each formulation wastested against five standard USP test organisms: S. aureus (SA, ATCC#6538), P. aeruginosa (PA, ATCC #9027), E. coli (EC, ATCC #8739), C.albicans (CA, ATCC #10231), and A. niger (AN, ATCC #16404). Formulationswere inoculated with suspensions of each organism in an amount thatrecovers less than 100 CFU (colony forming units) per filter.Inoculations for each formulation were conducted in triplicate.

Test samples were stored at 20-25° C. for the duration of the study.Testing was performed at Time 0, 24, and 48 hours after inoculation. Atappropriate time points, the samples were filtered through a 0.45micrometer membrane filter and washed with 0.1% sterile peptone solutionto remove the preservatives for the purpose of recovering themicroorganisms. The washed filters were aseptically removed and placedon appropriate microbiological growth medium.

S. aureus, P. aeruginosa and E. coli were plated on tripticase soy agarand incubated aerobically at 30-35° C. for 2 days or until countablecolonies were formed, whichever occurred first. C. albicans and A. nigerwere plated on sabouraud dextrose agar and aerobically incubated at20-25° C. for 7 days or until countable colonies were formed, whicheveroccurred first.

Microbial inhibition was considered resistant to microbial growth ifthere was a delay in onset or retardation of growth such that there wasless than 10-fold (1 log) increase in viable colonies over a 24-hourperiod.

The effectiveness of EDTA alone or in combination with benzyl alcohol onin microbial inhibition over a 24 hour period is demonstrated in Table 2below. From these experiments it can be concluded that the presence ofEDTA and benzyl alcohol either alone or in combination demonstratedmicrobial retardation capabilities.

TABLE 2 Microbial Retardant (% Time Formulation w/v) Point EC SA PA CAAN Example 1 None  0 21 32 7 29 14 24 hours TNTC >300 210 TNTC 19 48hours C TNTC C C 35 Example 2 0.005% EDTA  0 30 24 11 35 36 24 hours 221 2 233 30 48 hours 0.3 14 5 310 25 Example 3 0.01% EDTA  0 23 5 5 2031 24 hours 4 15 0.1 77 17 48 hours 3 16 0 250 40 Example 4 0.4% Benzyl 0 26 21 17 22 16 Alcohol 24 hours 63 34 2 45 19 48 hours 102 66 1 91037 Example 5 0.1% Benzyl  0 26 19 15 34 38 Alcohol/0.005% 24 hours 0.310 0.3 300 36 EDTA 48 hours 2 5 0 370 51 TNTC = too numerous to count C= Confluent growth

Example 2

Formulations containing antimicrobial agents as shown in Table 3 belowwere prepared by adding stock solutions of anti microbial agent atappropriate concentration to clevidipine emulsions.

TABLE 3 (% w/v) Function Example 6 Example 7 Active ingredientClevidipine butyrate 0.05 0.05 Oil phase Soybean oil 20 20 IsotonicityGlycerin 2.25 2.25 adjuster Microbial Growth Disodium Edetate 0.0050.005 Retardant Microbial Growth Sodium Citrate 0.1 0 RetardantAntioxidant Sodium Ascorbate 0 0.1 Emulsifier Purified egg yolk 1.2 1.2phospholipids pH adjuster Sodium hydroxide q.s q.s Aqueous Phase Waterfor Injection q.s to 100% q.s. to 100%

Compositions described above were evaluated for microbial growthfollowing the procedure described previously. The effectiveness of EDTAin combination with sodium citrate or sodium ascorbate in retardingmicrobial growth over a 24 hour period is demonstrated in Table 4 below.From these experiments it can be concluded that the presence of EDTA incombination with sodium citrate or sodium ascorbate demonstratedmicrobial retardation capabilities.

TABLE 4 Microbial Time Formulation Retardant (% w/v) Point EC SA PA CAAN Example 1 None  0 21 32 7 29 14 24 hours TNTC >300 210 TNTC 19 48hours C TNTC C C 35 Example 6 0.005% EDTA/  0 19 14 9 25 31 0.1% Sodium24 hours 2 5 4 55 39 Citrate 48 hours 0 3 23 85 34 Example 7 0.005%EDTA/  0 25 30 16 29 34 0.1% Sodium 24 hours 0 25 26 209 37 Ascorbate 48hours 0 19 42 3 > 300 45 TNTC = too numerous to count C = Confluentgrowth

Example 3

Clevidipine emulsion formulations were exposed to high intensity light(combination of cool white fluorescent and near UV light) in an effortto demonstrate the impact of light on the chemical stability and theresults are shown in Table 5 below:

TABLE 5 Oxidative Degradants Microbial Light Exposure RetardantAntioxidant (1.2 Million Lux Hours) Formulation (% w/v) (% w/v) H324/78H152/66 Example 1 None None 0.2 0.4 Example 2 0.005% EDTA None 0.2 0.2Example 7 0.005% EDTA 0.1% Sodium <0.1 Not Ascorbate Detected

It was unexpectedly found that the addition of ascorbic acid in thepresence of EDTA was effective in inhibition of photodegradation ofclevidipine in the emulsion, without counteracting the antimicrobialeffectiveness of EDTA.

Example 4

Clevidipine emulsion formulations with EDTA alone or in combination withsodium citrate were prepared by adding EDTA and/or sodium citrate as apart of the emulsion process in order to confirm the antimicrobialcharacteristics previously observed when added as a solution to aprepared emulsion. The emulsion formulations shown below were preparedas follows: The oil phase containing soybean oil and egg yolkphospholipids was mixed with the aqueous phase containing glycerin,disodium edetate with or without sodium citrate and water atapproximately 70° C. to form a coarse emulsion. The pH of the coarseemulsion was adjusted using sodium hydroxide. Following pH adjustment,the coarse emulsion was homogenized under high pressure to produce afine particle size and thus a stable emulsion. The emulsion was filledinto appropriate containers and sterilized in an autoclave.

These formulations were evaluated for microbial growth inhibition andthe results are shown in Table 6 below.

TABLE 6 Microbial Retardant (% Time Formulation w/v) Point EC SA PA CAAN Example 2 0.005% EDTA  0 25 37 27 24 37 24 hours 1 30 2 75 44 48hours 1 22 1 99 50 Example 8 0.025% EDTA  0 29 42 23 26 35 24 hours 1 381 82 32 48 hours 1 26 2 112 30 Example 9 0.001% EDTA  0 29 41 15 29 4024 hours 2 42 0 115 39 48 hours 1 26 0 133 39 Example 10 0.0025% EDTA/ 0 30 45 7 29 38 0.02% Sodium 24 hours 2 32 2 68 37 Citrate 48 hours 129 0 85 88 Example 11 0.001% EDTA/  0 26 32 29 22 40 0.02% Sodium 24hours 1 39 0 64 41 Citrate 48 hours 1 26 7 40 41

The results demonstrate that varying concentrations of EDTA either aloneor in combination with sodium citrate were effective in retardingmicrobial growth when added as a part of the manufacturing process.

Example 5

The physical stability of emulsions with anti microbial agents wasevaluated via zeta potential measurement. The zeta potential, which is ameasure of charge around the oil droplet, was determined to evaluate thephysical stability of emulsion formulations containing antimicrobialagents. A larger absolute value of zeta potential results in increasedelectrostatic repulsion between oil droplets and is indicative ofenhanced physical stability. The results demonstrated that the absolutevalue of zeta potential decreased in presence of EDTA alone or incombination with sodium citrate which was surprising given that theirconcentrations were low.

In an attempt to improve the physical stability of emulsions, use ofoleic acid a co-emulsifier was investigated. Clevidipine emulsion witholeic acid as shown in the example below was manufactured using thefollowing process: The oil phase containing soybean oil, clevidipine,egg yolk phospholipids and oleic acid was mixed with the aqueous phasecontaining glycerin, disodium edetate and water at approximately 70° C.to form a coarse emulsion. The pH of the coarse emulsion was adjustedusing sodium hydroxide. Following pH adjustment, the coarse emulsion washomogenized under high pressure to produce a fine particle size and thusa stable emulsion. The emulsion was filled into appropriate containersand sterilized in an autoclave.

TABLE 7 % w/v Function Example 12 Active ingredient Clevidipine butyrate0.05 Oil phase Soybean oil 20 Isotonicity Glycerin 2.25 adjusterMicrobial Growth Disodium Edetate 0.005 Retardant Coemulsifier Oleicacid 0.03 Emulsifier Purified egg yolk 1.2 phospholipids pH adjusterSodium hydroxide qs Aqueous Phase Water for Injection qs to 100%

The above composition was evaluated for zeta potential and microbialgrowth inhibition.

Inhibition of microbial growth for the above Example is shown in Table 8below for 12, 24, and 30 hours.

TABLE 8 CFU/5 mL Challenge Organism 0 Hours 12 Hours 24 Hours 30 HoursS. marsacens (SM) 29 32 32 40 Staph. epidermidis (SE) 30 6 5 5

The results for zeta potential are shown in Table 9 below anddemonstrate that inclusion of oleic acid at a low concentrationunexpectedly showed a substantial increase in zeta potential andphysical stability of the emulsion compared to emulsions comprising EDTAalone or in combination with sodium citrate.

TABLE 9 Microbial Retardant Co-emulsifler Zeta Potential Formulation (%w/v) (% w/v) (mV) pH Example 1 None None −25.6 7.7 Example 2 0.005% EDTANone −24.8 7.7 (Lab scale) Example 2 0.005% EDTA None −17.6 7.7 (pilotscale) Example 11 0.001% EDTA/ None −18.3 7.4 0.02% SC Example 12 0.005%EDTA 0.03% −36.1 8.0 Oleic acid

In addition as demonstrated in Table 10 below, oleic acid did not impactthe antimicrobial effectiveness of EDTA.

TABLE 10 Microbial Retardant Time Formulation (% w/v) Point EC SA PA CAAN Example 2 0.005% EDTA  0 25 37 27 24 37 24 hours 1 30 2 75 44 48hours 1 22 1 99 50 Example 11 0.005% EDTA/  0 39 32 35 70 50 0.03% OleicAcid 24 hours 1 49 49 305 75 48 hours 0 41 70 395 40

The invention claimed is:
 1. A pharmaceutical formulation comprising (a)an effective amount of clevidipine, or a pharmaceutically acceptablesalt or ester, (b) an antimicrobial agent, EDTA, present at about 0.001to about 1.5% w/v, (c) a lipid, (d) an emulsifier, (e) a tonicitymodifier, and (f) water wherein the formulation is resistant tomicrobial growth.
 2. The pharmaceutical formulation of claim 1, whereinEDTA is present at about 0.001 to about 0.025% w/v.
 3. Thepharmaceutical formulation of claim 1, wherein the lipid is selectedfrom the group consisting of soybean oil, safflower seed oil, olive oil,cottonseed oil, sunflower oil, sesame oil, peanut oil, corn oil, mediumchain triglycerides, triacetin, propylene glycol diesters,monoglycerides, and a mixture of two or more thereof.
 4. Thepharmaceutical formulation of claim 1, wherein the emulsifier isselected from the group consisting of egg yolk phospholipids, soybeanphospholipids, synthetic phosphatidyl cholines, purified phosphatidylcholines and hydrogenated phosphatidyl choline, and mixtures of two ormore thereof.
 5. The pharmaceutical formulation of claim 1, furthercomprising an antioxidant selected from the group consisting of sodiumascorbate, sodium citrate, cysteine hydrochloride, sodium bisulfate,sodium metabisulfite, sodium sulfite ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate,tocopherol, and a pharmaceutically acceptable salt thereof.
 6. Thepharmaceutical formulation of claim 1 having a pH of about 6.0 to about8.8.
 7. The pharmaceutical formulation of claim 2, further comprising aco-emulsifier wherein the co-emulsifier is selected from the groupconsisting of glycerin, poloxamers, polyoxyethylene castor oilderivative, poloxamines, polyoxyethylene stearates, polyoxyethylenesorbitan fatty acid esters, sorbitan fatty acid esters, polysorbates,tocopherol PEG succinate, cholic acid, deoxycholic acid, oleic acid, andpharmaceutically acceptable salts thereof.
 8. The formulation of claim 1wherein microbial growth is delayed or retarded such that there is lessthan 10-fold (1 log) increase in viable microbial colonies over a24-hour period.